scholarly journals PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo

Reproduction ◽  
2004 ◽  
Vol 127 (6) ◽  
pp. 653-667 ◽  
Author(s):  
Judith J Eckert ◽  
Amanda McCallum ◽  
Andrew Mears ◽  
Martin G Rumsby ◽  
Iain T Cameron ◽  
...  
Keyword(s):  
Tight Junction ◽  
Normal Development ◽  
Epithelial Differentiation ◽  
Cell Contact ◽  
Contact Pattern ◽  
Mouse Blastocyst ◽  
Membrane Assembly ◽  
Kinase C ◽  
Protein Membrane ◽  
Pkc Activation

Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1α+), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPA-mediated PKC activation stimulates only that of ZO-1α+. The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1α+ membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKCζ) and we find that both PKCδ and ζ relocate upon activator treatment to colocalise partially with the TJ proteins in isolated ICMs, we suggest that at least PKCδ and ζ may play a central role in regulating TJ membrane assembly.

scholarly journals 103 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IS DISPENSIBLE DURING REGULATION OF TIGHT JUNCTION MEMBRANE ASSEMBLY BY CELL CONTACT PATTERN AND PKC SIGNALING

2005 ◽  
Vol 17 (2) ◽  
pp. 202
Author(s):  
J. Eckert ◽  
A. Mears ◽  
I. Cameron ◽  
T. Fleming
Keyword(s):  
Tight Junction ◽  
Intercellular Communication ◽  
Cell Contact ◽  
Contact Pattern ◽  
Gap Junctional Intercellular Communication ◽  
Membrane Assembly ◽  
Junctional Proteins ◽  
Pkc Signaling ◽  
Gap Junctional

Contact symmetries are involved in regulating cell lineage segregation during blastocyst biogenesis when tight junction (TJ) membrane assembly is restricted to the epithelial trophectoderm (TE). Manipulation of cell contact patterns by immunosurgical isolation of inner cell masses (ICMs) providing a contact-free cell surface serves as a switch to induce TE differentiation upon in vitro culture. In this model, protein kinase C (PKC)-mediated signaling up-regulates TJ membrane assembly. Whether signaling via gap junctional intercellular communication (GJIC) affects these processes is controversial. The current study investigates the interrelationship between changes in cell contact pattern, PKC signaling, and GJIC on TE differentiation and TJ assembly. Eight-cell embryos flushed from MF1 mice were cultured in T6/BSA to time development to early blastocyst stage (<2 h of cavitation). Laser confocal microscopy (BioRad MRC 600, BioRad Laboratories, Inc., Hertfordshire, UK) after immunostaining with antibodies against PKCδ, θ, λ, or ζ isoforms (Transduction Labs, Oxford, UK or Sigma) or junctional proteins (E-cadherin, ZO-2, Occludin, ZO-1α+, Desmoplakin) combined with ALEXA 488 conjugated secondary antibodies (Cambridge Bioscience, Oxford, UK) was used to determine the distribution of PKCs and junctional proteins in intact blastocysts and fully and partially isolated ICMs after immunosurgery and in vitro culture in DMEM + 10% FCS (Eckert et al. 2004 Reproduction 127, 653). While broad PKC activators (1 μM 12-O-tetradecanoylphorbol-13-acetate or Indolactam; Calbiochem, Nottingham, UK) accelerate membrane assembly of the TJ proteins ZO-2 and ZO-1α+ in fully isolated ICMs this up-regulation was suppressed in intact blastocysts (n = 32–47 per treatment and antibody) and in partially isolated ICMs (remnants of lysed TE remaining surrounding the ICM; n = 17–21 per treatment and antibody) for up to 24 h with no TJ protein detectable within the ICM, even after two consecutive rounds of TE lysis (n = 13–22 per treatment and antibody). When GJIC was inhibited during blastocyst formation in vitro and in cultured fully isolatd ICMs by 18 α-glycyrrhetinic acid (AGA, 65 μM; Sigma), cavitation rate and distribution of PKCs or junction assembly were not affected compared to controls (70–80% cavitated with characteristic distribution of junctional proteins and PKCs; P > 0.05, ANOVA; n = 15–20 per treatment, antibody, and cell contact pattern). When GJIC inhibition by AGA was confirmed by Lucifer yellow (Sigma) injection (no dye transfer in 82–100%, n = 14–17 per contact pattern), GJIC was also absent in 50% of fully isolated ICMs without AGA treatment, suggesting that cell contact modulation may affect GJIC. Taken together, our data suggest that cell contact pattern regulates TJ assembly via PKC signaling pathways and may also affect GJIC. GJIC appeared dispensable during cavitation, TJ assembly, and PKC signaling. A better understanding of the interrelationships between different signaling mechanisms may help to improve embryo culture methods and viability. Funding by the Wellcome Trust and MRC is gratefully acknowledged.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux

2019 ◽  
Vol 166 (4) ◽  
pp. 309-315 ◽  
Author(s):  
Taro Watanabe ◽  
Noriyuki Kioka ◽  
Kazumitsu Ueda ◽  
Michinori Matsuo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Translational Regulation ◽  
Cholesterol Efflux ◽  
Degradation Rate ◽  
Active Form ◽  
Density Lipoprotein ◽  
Protein Levels ◽  
Kinase C ◽  
Pkc Activation

Abstract ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

scholarly journals Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading.

1993 ◽  
Vol 4 (3) ◽  
pp. 271-281 ◽  
Author(s):  
J S Chun ◽  
B S Jacobson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Hela Cell ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Subsequent Formation ◽  
Sequence Of Events ◽  
Kinase C ◽  
Pkc Activation

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

scholarly journals Potassium channels modulate canine pulmonary vasoreactivity to protein kinase C activation

1999 ◽  
Vol 277 (3) ◽  
pp. L558-L565 ◽  
Author(s):  
Scott A. Barman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Channel Blocker ◽  
Pressor Response ◽  
Vascular Occlusion ◽  
Membrane Depolarization ◽  
Delayed Rectifier ◽  
Vascular Compliance ◽  
Kinase C ◽  
Pkc Activation

The role of Ca2+-activated K+-channel, ATP-sensitive K+-channel, and delayed rectifier K+-channel modulation in the canine pulmonary vascular response to protein kinase C (PKC) activation was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. The PKC activators phorbol 12-myristate 13-acetate (PMA; 10−7 M) and thymeleatoxin (THX; 10−7 M) significantly increased pulmonary arterial and pulmonary venous resistances and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. The Ca2+-activated K+-channel blocker tetraethylammonium ions (1 mM), the ATP-sensitive K+-channel inhibitor glibenclamide (10−5 M), and the delayed rectifier K+-channel blocker 4-aminopyridine (10−4 M) potentiated the pressor response to both PMA and THX on the arterial and venous segments and also further decreased pulmonary vascular compliance. In contrast, the ATP-sensitive K+-channel opener cromakalim (10−5 M) attenuated the vasoconstrictor effect of PMA and THX on both the arterial and venous vessels. In addition, membrane depolarization by 30 mM KCl elicited an increase in the pressor response to PMA. These results indicate that pharmacological activation of PKC elicits pulmonary vasoconstriction. Closure of the Ca2+-activated K+ channels, ATP-sensitive K+ channels, and delayed rectifier K+ channels as well as direct membrane depolarization by KCl potentiated the response to PMA and THX, indicating that K+ channels modulate the canine pulmonary vasoconstrictor response to PKC activation.

Adenosine-stimulated Ca2+reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system

1998 ◽  
Vol 274 (4) ◽  
pp. F736-F743 ◽  
Author(s):  
Joost G. J. Hoenderop ◽  
Anita Hartog ◽  
Peter H. G. M. Willems ◽  
René J. M. Bindels
Keyword(s):  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Concomitant Increase ◽  
Cgs 21680 ◽  
Kinase C ◽  
Intracellular Ca ◽  
Collecting Duct Cells ◽  
Camp Formation ◽  
Pkc Activation ◽  
Permeable Supports

Confluent monolayers of immunodissected rabbit connecting tubule and cortical collecting duct cells, cultured on permeable supports, were used to study the effect of adenosine on net apical-to-basolateral Ca2+ transport. Apical, but not basolateral, adenosine increased this transport dose dependently from 48 ± 3 to 110 ± 4 nmol ⋅ h−1 ⋅ cm−2. Although a concomitant increase in cAMP formation suggested the involvement of an A2 receptor, the A2 agonist CGS-21680 did not stimulate Ca2+ transport, while readily increasing cAMP. By contrast, the A1 agonist N 6-cyclopentyladenosine (CPA) maximally stimulated Ca2+transport without significantly affecting cAMP. Adenosine-stimulated transport was effectively inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopenthylxanthine but not the A2 antagonist 3,7-dimethyl-1-propargylxanthine, providing additional evidence for the involvement of an A1 receptor. Both abolishment of the adenosine-induced transient increase in intracellular Ca2+ concentration by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid and downregulation of protein kinase C (PKC) by prolonged phorbol ester treatment were without effect on adenosine-stimulated Ca2+ transport. The data presented suggest that adenosine interacts with an apical A1 receptor to stimulate Ca2+ transport via a hitherto unknown pathway that does not involve cAMP formation, PKC activation, and/or Ca2+ mobilization.

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